Wnt signalling is essential for regulation of embryonic development and adult tissue homeostasis1,2,3, and aberrant Wnt signalling is frequently associated with cancers4. Wnt signalling requires palmitoleoylation on a hairpin 2 motif by the endoplasmic reticulum-resident membrane-bound O-acyltransferase Porcupine5,6,7 (PORCN). This modification is indispensable for Wnt binding to its receptor Frizzled, which triggers signalling8,9. Here we report four cryo-electron microscopy structures of human PORCN: the complex with the palmitoleoyl-coenzyme A (palmitoleoyl-CoA) substrate; the complex with the PORCN inhibitor LGK974, an anti-cancer drug currently in clinical trials10; the complex with LGK974 and WNT3A hairpin 2 (WNT3Ap); and the complex with a synthetic palmitoleoylated WNT3Ap analogue. The structures reveal that hairpin 2 of WNT3A, which is well conserved in all Wnt ligands, inserts into PORCN from the lumenal side, and the palmitoleoyl-CoA accesses the enzyme from the cytosolic side. The catalytic histidine triggers the transfer of the unsaturated palmitoleoyl group to the target serine on the Wnt hairpin 2, facilitated by the proximity of the two substrates. The inhibitor-bound structure shows that LGK974 occupies the palmitoleoyl-CoA binding site to prevent the reaction. Thus, this work provides a mechanism for Wnt acylation and advances the development of PORCN inhibitors for cancer treatment.
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Mechanism of action for small-molecule inhibitors of triacylglycerol synthesis
Nature Communications Open Access 29 May 2023
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The 3D cryo-EM maps have been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-26707, EMD-26708, EMD-26709, EMD-26710 and EMD-26711. Atomic coordinates for the atomic model have been deposited in the Protein Data Bank under the accession numbers 7URA, 7URC, 7URD, 7URE and 7URF. Additional data supporting the findings in this study are provided as source data and supplementary information to this manuscript. Source data are provided with this paper.
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Cryo-EM data were collected at the UT Southwestern Medical Center Cryo-EM Facility (funded in part by the CPRIT Award RP170644). We thank D. Stoddard, L. Friedberg, L. Esparza and Y. Qin for technical support; A. Lemoff at the UTSW Proteomics Core for mass spectrometry analysis; C. Chattergee and Z. Chen for discussion; and E. Debler and P. Schmiege for editing the manuscript. This work was supported by NIH P01 HL020948, P01 HL160487, R01 GM135343 and Welch Foundation (I-1957) (to X.L.). N.E.-M. was funded in part by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy—EXC 2008-390540038-UniSysCat. X.Q. is a recipient of a DDBrown Fellowship of Life Sciences Research Foundation. B.W. is a Southwestern Medical Foundation Scholar in Biomedical Research. X.L. is a Damon Runyon-Rachleff Innovator supported by the Damon Runyon Cancer Research Foundation (DRR-53S-19) and a Rita C. and William P. Clements Jr Scholar in Biomedical Research at UT Southwestern Medical Center.
The authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 LC-MS analysis of PORCN-mediated WNT3Ap acylation and MS analysis of pamWNT3Ap.
a, The secretion pathway of WNT ligand. In the endoplasmic reticulum (yellow), WNT (purple) is modified by PORCN (orange). Transport of the modified WNT is mediated by WLS (blue). WNTs are secreted to extracellular space via vesicles. After binding to FZD (green), WNTs trigger the signal transduction. Notum (light blue) acts as a deacylase, removing the lipid of WNT to abolish the signal. The structure of palmitoleated WNT3A hairpin 2 is shown. b, Representative LC chromatograms of PORCN-mediated WNT3Ap acylation with (red) and without (blue) LGK974. ESI-MS: m/z calculated for [M+3H]3+ 840.041, found 840.039, and m/z calculated for [palmitoleated WNT3Ap+3H]3+ 918.779, found 918.778. c, Quantitative analysis of the product ratio after the reaction. Data are mean ± s.d. (n = 3 biologically independent experiments). ****P ≤ 0.0001, two-tailed unpaired t-test using GraphPad Prism 8. d, LC analysis of pamWNT3Ap. The samples (panels b and d) were applied to a HPLC column (DiscoveryBIO wide pore C5, 4.6x150mm, 5 μm) using a gradient in which percentage of solvent B increases from 20% to 80% in 6 min at 1.5 mL/min. e, Mass spectrometry analysis of [pamWNT3Ap+3H]3+. ESI-MS: m/z calculated for [M+3H]3+ (C124H196N33O28S5) 918.451, found 918.446
Extended Data Fig. 2 Protein Purification and data processing of palmitoleoyl-CoA-bound and LGK974-bound PORCN.
a, Representative Superose 6 increase 10/300 GL gel-filtration chromatogram of PORCN complex with Fab2C11. The peak fraction is shown on SDS-PAGE with molecular markers. Each protein is indicated. For gel source data, see Supplementary Fig. 1a. b, The data processing of palmitoleoyl-CoA-bound PORCN. The cryo-EM 3D classes as well as the mask used for the refinement are shown. The final cryo-EM map after cryoSPARC refinement was sharpened with a B-factor value of −170 Å2. c, Fourier shell correlation (FSC) curve of palmitoleoyl-CoA-bound PORCN map as a function of resolution using cryoSPARC output. d, The data processing of LGK974-bound PORCN. The cryo-EM 3D classes as well as the mask used for the refinement are shown. The final cryo-EM map after cryoSPARC refinement was sharpened with a B-factor value of −130 Å2. e, Fourier shell correlation (FSC) curve of LGK974-bound PORCN map as a function of resolution using cryoSPARC output.
Extended Data Fig. 3 cryo-EM map of structural elements of palmitoleoyl-CoA-bound and LGK974-bound PORCN.
a, The Fourier shell correlation (FSC) curves calculated between the refined structure model and the half map used for refinement (yellow), the other half map (gray) and the full map (blue) of palmitoleoyl-CoA-bound PORCN. b, Density map colored by local resolution estimation using cryoSPARC. c, The major helices of PORCN. d, MD simulation suggests that palmitoleoyl-CoA binds to PORCN in the curled-up conformation. After 100 ns simulations, the interaction between residues W300, H357 and the curled-down palmitoleoyl-CoA disrupts (the right bottom panel). The palmitoleoyl-CoA in either conformation with the cryo-EM map is shown. e, The Fourier shell correlation (FSC) curves calculated between the refined structure model and the half map used for refinement (yellow), the other half map (gray) and the full map (blue) of palmitoleoylated WNT3A-bound PORCN. f, Density map colored by local resolution estimation using cryoSPARC. g, The major helices of PORCN.
Extended Data Fig. 4 Data processing of WNT3Ap/LGK974-bound PORCN and pamWNT3Ap-bound PORCN.
a, The data processing of WNT3Ap/LGK974-bound PORCN. The cryo-EM 3D classes as well as the mask used for the refinement are shown. The final cryo-EM map after cryoSPARC refinement was sharpened with a B-factor value of −147 Å2. b, Fourier shell correlation (FSC) curve of WNT3Ap/LGK974-bound PORCN map as a function of resolution using cryoSPARC output. c, The data processing of pamWNT3Ap-bound PORCN. The cryo-EM 3D classes as well as the mask used for the refinement are shown. The final cryo-EM map after cryoSPARC refinement was sharpened with a B-factor value of −140 Å2. d, Fourier shell correlation (FSC) curve of pamWNT3Ap-bound PORCN map as a function of resolution using cryoSPARC output.
Extended Data Fig. 5 cryo-EM map of structural elements of WNT3Ap/LGK974-bound PORCN and pamWNT3Ap-bound PORCN.
a, The Fourier shell correlation (FSC) curves calculated between the refined structure model and the half map used for refinement (yellow), the other half map (gray) and the full map (blue) of WNT3Ap/LGK974-bound PORCN. b, Density map colored by local resolution estimation using cryoSPARC. c, The major helices of PORCN, WNT3Ap and LGK974. d, The Fourier shell correlation (FSC) curves calculated between the refined structure model and the half map used for refinement (yellow), the other half map (gray) and the full map (blue) of pamWNT3Ap-bound PORCN. e, Density map colored by local resolution estimation using cryoSPARC. f, The major helices of PORCN and pamWNT3Ap.
Extended Data Fig. 6 The structure of DHHC20 and the structural comparison of PORCN (gray) with the other MBOAT proteins.
a, The structure of DHHC20. The zinc ions are shown as gray balls. b, The structural comparison with DltB. c, The structural comparison with HHAT. IMP-1575, an HHAT inhibitor, is shown as green sticks. The catalytic H379 of HHAT is shown as sticks. d, The structural comparison with DGAT1. e, The structural comparison with ACAT1. The LGK974 is shown as magenta sticks. Nevanimibe, an ACAT1 inhibitor, is shown as green sticks. The acyl-CoA in the DGAT and ACAT1 is shown as yellow sticks.
Extended Data Fig. 7 Purification of human PORCN variants for activity assays.
Representative Superose 6 increase 10/300 GL gel-filtration chromatogram of PORCN variants in buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, and 0.06% Digitonin.
Extended Data Fig. 8 Superimposing the WNT8A-FZD and WNT8A-WLS complexes into the structure of pamWNT3Ap-bound PORCN and the model of WNT3A-bound PORCN.
a, Superimposing the hairpin 2 of the structure of WNT8A-FZD complex into PORCN-pamWNT3Ap complex. The clash between WNT8A ligand and PORCN is indicated by a dashed circle. b, Superimposing the hairpin 2 of the structure of WNT8A-WLS complex into PORCN-pamWNT3Ap complex. The clash between WNT8A ligand and PORCN is indicated by a dashed circle. PAM, palmitoleic acid (red sticks). c, The predicted structure of WNT3A-bound PORCN. Three hairpins of WNT3A have been indicated. d, Structural comparison of the predicted PORCN structure to the cryo-EM structure (PDB:7URA).
Extended Data Fig. 9 The structure of HHAT-SHH-N complex.
a, Representative Superose 6 increase 10/300 GL gel-filtration chromatogram of HHAT-SHH-N complex with Fab3H02. The peak fraction is shown on SDS-PAGE with molecular markers. Each protein is indicated. For gel source data, see Supplementary Fig. 1b. b, Fourier shell correlation (FSC) curve as a function of resolution using RELION-3 output. c, Cryo-EM map of the complex after 3D refinement reveals the mean body of SHH-N protein at the threshold level of 0.003 but not 0.01.
Supplementary Figure 1
This file contains the uncropped gels used in Extended Data Figs. 2a and 9a.
Supplementary Video 1
Molecular dynamics simulations of PORCN with palmitoleoyl-CoA in curled-up conformation in 100 ns time scale. The PORCN is colored in cyan, and the palmitoleoyl-CoA is colored in orange. Residues Trp300 and His357 are shown in sticks.
Supplementary Video 2
Molecular dynamics simulations of PORCN with palmitoleoyl-CoA in curled-down conformation in 100 ns time scale. The PORCN is colored in cyan, and the palmitoleoyl-CoA is colored in gray. Residues Trp300 and His357 are shown in sticks.
Supplementary Video 3
Molecular dynamics simulations of WNT3A-PORCN complex in 100 ns time scale. The PORCN is colored in cyan and the WNT3A is colored in blue. The disulfide bonds are shown in sticks.
Supplementary Video 4
Molecular dynamics simulations of PORCN in 100 ns time scale. The PORCN TM11 is colored in red.
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Liu, Y., Qi, X., Donnelly, L. et al. Mechanisms and inhibition of Porcupine-mediated Wnt acylation. Nature 607, 816–822 (2022). https://doi.org/10.1038/s41586-022-04952-2
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Mechanism of action for small-molecule inhibitors of triacylglycerol synthesis
Nature Communications (2023)
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